The experiments in this proposal are designed to investigate the signal transduction cascades that are triggered when mammalian cells are exposed to extracellular stimuli. Understanding how such signaling pathways work is important because in clinical disorders such as cancer, signal transduction pathways may be improperly regulated or constitutively activated. This project will focus on the analysis of signaling pathways that are mediated by mammalian serine/threonine kinases related to the yeast signaling enzyme STE20. Of particular interest will be PAK4, a new protein that we have identified, that is distantly related to the PAK family of protein kinases. The roles for these proteins will be investigated in cultured mammalian fibroblasts and macrophage cell lines. The following questions will be addressed: 1) Which MAP Kinase pathways are activated by PAK4 and other STE20 like kinases? This will involve transient transfection experiments in which expression vectors for wild-type or constitutively active mutants of STE20 related proteins are transfected into mammalian cells. They will be co-transfected with expression vectors for epitope tagged JNK, ERK, or p38 MAP Kinases. Immune complex kinase assays will be carried out to determine which MAP Kinase pathways become activated. Experiments will also be carried out to examine the mechanism of activation of these pathways. 2) How are PAK4 and other STE20 like kinases regulated by extracellular stimuli? Immune complex kinase assays will be used to examine the activation of STE20 related proteins in response to extracellular stimuli such as cytokines and growth factors. Signaling enzymes required for their activation will also be investigated. 3) Is PAK4 regulated by small GTP binding proteins of the Rho family? Sequence analysis suggests that PAK4 may be a new target for GTP binding proteins. Experiments will be designed to determine whether it interacts with, and is activated by, GTP binding proteins of the Rho family. 4) What are the direct downstream targets of PAK4 and other STE20 like kinases? Experiments are designed to determine whether STE20 related proteins directly activate known signaling enzymes such as MEKK. Experiments are also designed to identify novel substrates. This will involve using the yeast two hybrid system to identify interacting proteins, and screening expression libraries to identify direct targets.